A major barrier to improving cure rates in locally advanced cancers is our inability to make progress beyond what chemoradiation (CRT) can currently deliver. Combination strategies using molecular targeted therapies with CRT hold promise for improving outcomes further. While many drugs could enhance the effects of radiation alone, we have discovered that the effects are quite unpredictable when drugs are combined with chemotherapy and radiotherapy. The successful translation of adding molecular targeted agents to CRT would require an understanding of the molecular pathways that enable the cancer cell to survive under conditions of CRT. Inhibiting these pathways with molecular targeted drugs will be synergistic with CRT in the cancer-specific context. Using a set of molecular targeted drugs from the CTEP portfolio as an initial starting point, we will investigate two hard-to-treat cancer types treated with CRT, non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC). We will identify drugs that could synergize with radiation and CRT using a high throughput clonogenic survival screen that we have developed on validated cancer lines and then test the most clinically promising combinations of agents to multiple cell lines with varying genetic backgrounds, first in vitro and then further validated using 2 in vivo models: a panel of patient-derived xenografts (PDXs) and orthotopic tumor models using syngeneic tumors, all done in combination with clinically-relevant chemotherapies. The pharmacokinetic and pharmacodynamic properties of these drugs with chemotherapy in animals and tumors will be assessed in order to determine the optimal sequencing approach with conventionally fractionated radiotherapy. Since we have discovered that chemotherapy significantly alters the response of cancer cells to radiation and targeted drugs, we will also evaluate the molecular mechanisms that explain the response to CRT, and identify potential factors that may influence this response using 4 major approaches. In the first more classic approach, we will assess DNA damage repair pathways and reactive oxygen species generation when targeted agents are combined with radiation or CRT. Second, we will use reverse phase protein arrays (RPPA) to assess the functional proteome to determine pathways that may be altered with molecular targeted drugs in the setting of RT or CRT. In the third approach, we will use Stable Isotope Labeling with Amino Acids (SILAC) to assess global proteomic and phosphoproteomic changes that occur with radiation and CRT treatment, and how these pathways could be altered with specific molecular targeted therapies. Lastly, we will use Imaging Mass Spectrometry to analyze drug distribution within the various tumor models and assess how the pharmacodynamic heterogeneity impacts CRT responsiveness. Our proposal will not only identify the most promising drugs that could best be combined with CRT in NSCLC and PDAC, but we will have identified molecular and tumor factors that confer drug resistance which will enable future development of novel targeted strategies to enhance CRT or appropriately select patient for personalized therapy. Our approach will generate the high quality preclinical data and novel insights to fulfill the overall FOA objective, which is ?to accelerate the pace at which combined modality treatments with greater efficacy are identified and incorporated into standard practices for treatments?.